Cilia are thread-like microtubule-based cell extensions which function in cell locomotion, fluid transport, and signaling. Many developmental disorders and diseases are caused by defects in ciliary function and assembly. To assemble cilia of a specific size and composition, cells have to transport hundreds of different proteins from the cell body into the organelle. Intraflagellar transport (IFT), a bidirectional motility of protein particles along ciliary microtubules, is assumed to be the major pathway for protein transport in cilia. IFT is required for ciliary assembly, maintenance, and signaling, however, it remains largely unknown which proteins are transported by IFT. It is also unclear where in the cilium cargoes are unloaded from IFT and whether the amount of protein transported by IFT is regulated. Because ciliary proteins are likely to be transported as single molecules or in small clusters, the analysis of their transport requires a highly sensitive imaging technique. Using Total Internal Reflection Fluorescence (TIRF) microscopy, we have established in vivo imaging of protein transport by IFT in cilia. We will analyze protein transport in cilia using the unicelluar model Chlamydomonas reinhardtii, which allows us to combine high resolution imaging in cilia with genetic manipulation and biochemical analysis of the organelle. We performed a comprehensive analysis of ciliary transport of the axonemal protein DRC4 and showed that DRC4-GFP depends on IFT for ciliary entry and distribution along the organelle. In Specific Aim 1, we will image distinct proteins selected from different ciliary compartments and substructures to determine how they interact with IFT to move into cilia. We will address the question of how IFT particles serve as carriers for many distinct proteins and how IFT transports proteins in the correct ratio into the organelle. We will test whether protein loading onto IFT particles depends on protein supply in the cell body and to which extent unloading of cargoes from IFT is spatially controlled. Our data show that the transport frequency of DRC4 is greatly increased when cilia grow, suggesting that the capacity of the IFT pathway can be modulated. The regulation of IFT is the focus of Specific Aim 2. We will analyze whether IFT particles isolated from growing and steady-state cilia are biochemically distinct and how cargo transport is affected in IFT mutants with small defects in the particle. The control of cargo influx is likely to be a prerequisite to establish a specific length of cilia, which is critical for its motile and signaling functions. We ill analyze IFT and cargo transport in mutants with defects in ciliary length regulation such as long flagella 2 (lf2). LF2 encodes a widely conserved CDK-like kinase with an emerging role in disease. IFT is disturbed in lf2 cilia; we will test the hypothesis that LF2 kinase is a regulator f IFT, which when defective results in overloading of IFT particles. We noted that IFT proteins accumulate in mutants with structural defects in cilia, which might indicate a feedback mechanism on the IFT pathway which alerts the cell of incorrectly assembled cilia. We will test whether cells use the IFT pathway to monitor the correct size and structure of cilia.